Synaptic vesicular Zn2+ has been regarded as a neuronal signaling modulator, thus my long-term goal is to study how the vesicular Zn2+ regulates brain function and to identify the mechanism by which synaptic vesicular Zn2+ dyshomeostasis is involved in neurodegeneration and brain injury. Monitoring the synaptic vesicular Zn2+ in neurons is of critical significance for achieving this goal. Currently, small molecule Zn2+ sensors were used to visualize vesicular Zn2+; however the small molecule sensors are limited by their nonspecific localizations and inability for long-term imaging. The major objectives of my proposed research are to generate biological imaging systems that can monitor vesicular Zn2+ dynamics in living neurons and animals with high spatio-temporal fidelity. The proposed biological imaging systems are exploiting the capability of genetically encoded sensors for specific targeting (specific subcelluar locations in specialized groups of cells) and long-term imaging. A novel single fluorescent protein (single-FP) based genetically encoded Zn2+ sensors will be developed and targeted into synaptic vesicles in neurons. In the preliminary studies, the prototype single-FP Zn2+ sensors were generated by attaching two zinc fingers of transcription factor Zap1 (ZF1 and ZF2) to the two ends of circularly permuted fluorescent protein (FP). When Zn2+ is bound, the formation of two zinc finger folds would cause the finger-finger interaction, which would induce subsequent conformational change of FP and the changes of fluorescent intensities. In the mentored phase of proposed research, the prototype single-FP sensors will be optimized for better fluorescent signals using cell- based screening of mutated sensor library. The validated single-FP Zn2+ sensors will then be incorporated into the synaptic vesicles, which will then be introduced into cultured neurons and zebrafish, generating cell-based and animal-based imaging systems. In the independent phase, both imaging systems will be evaluated and applied to biological studies. I will test a specific hypothesis: synaptic vesicular Zn2+ transporter ZnT3 utilizes a Zn2+/proton exchange mechanism to concentrate Zn2+ into vesicles during ischemia/reperfusion. In addition, I will explore the roles of synaptic vesicular Zn2+ in ischemic brain damage in zebrafish. These studies would not only verify the practicability of the imaging systems, but also discover the regulation mechanism of synaptic vesicular Zn2+ and their specific effects on neuronal recovery during brain ischemia/reperfusion in intact living animals. In conclusion, the proposed research will develop new imaging tools for monitoring synaptic vesicular Zn2+ in living neurons and animals with high spatial and temporal fidelity, which will offer a new method to study the signaling function of synaptic vesicular Zn2+. Additionally, utilization of these imaging systems in the ischemia models could elucidate how to modulate the synaptic vesicular Zn2+ for neuronal recovery during ischemia/reperfusion.